Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro

Jun Jie Tan,David J Milan,Kenji Miki,Tong Wu,Liye Zhu,King-Hwa Ling,Jacques P Guyette,Ling Xiao,Harald C Ott,Katrina J Hansen,Gurbani Kaur

doi:10.1038/s41467-021-24921-z

Abstract

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.

Highlights

Epicardial formation is necessary for normal myocardial morphogenesis
We build upon our previous work in cardiac tissue formation to demonstrate a simple method to generate preepicardial cells (PECs) from hiPSCs4,30, a premature form of epicardial cells expressing typical epicardial genes WT1, TBX18, SEMA3D, and SCX but capable of developing further to more mature epicardial cells after being in contact with CMs and recapitulating the developmental roles in embryonic heart formation, including epithelial-mesenchymal transition (EMT) and derivation of fibroblast and smooth muscle cells, and stimulation of ventricular myocyte proliferation partly via retinoic acid (RA)-dependent IGF signaling of CM proliferation
PECs and CMs have been shown to share a similar pool of cardiac precursors derived from LPM15,29, and lateral plate mesoderm (LPM) can be derived from human-induced pluripotent stem cells (hiPSCs) with prolonged CHIR treatment[8]

Summary

Introduction

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. We build upon our previous work in cardiac tissue formation to demonstrate a simple method to generate preepicardial cells (PECs) from hiPSCs4,30, a premature form of epicardial cells expressing typical epicardial genes WT1, TBX18, SEMA3D, and SCX but capable of developing further to more mature epicardial cells (upregulated additional markers UPK1B, ITGA4, ALDH1A2) after being in contact with CMs and recapitulating the developmental roles in embryonic heart formation, including EMT and derivation of fibroblast and smooth muscle cells, and stimulation of ventricular myocyte proliferation partly via RA-dependent IGF signaling of CM proliferation. In a facile method to generate and observe interactions in three-dimensional constructs, spheroid co-culture allows for the generation of electrically active cardiac-microtissue constructs with distinct luminal structures

Methods

Results

Conclusion